The immunomodulatory proteins, thymopoietin and thysplenin (formerly referred to as "splenin"), have been isolated from bovine and human thymus and spleen, respectively. Additionally, small peptides have been chemically synthesized which mimic the biological activity of thymopoietin and have been further modified to be provided with additional attributes such as resistance to enzymatic action. See, e.g. U.S. Pat. No. 4,505,853.
A large body of articles and patents have now been published relating to such proteins and synthesized peptides. U.S. Pat. No. 4,190,646 discloses the pentapeptide thymopentin which is the active site of thymopoietin and has the sequence Arg-Lys-Asp-Val-Tyr, as well as peptide compositions in which various groups are substituted onto the amino and/or carboxy termini of this pentapeptide. Both thymopoietin and thymopentin induce biological changes in two human T cell lines, CEM and MOLT-4, thereby indicating a role in stimulating biological activities of T cells. No analogs of thymopentin shorter than pentapeptides (5 amino acids in sequence) were found to be active on CEM cells.
Applicants' copending U.S. patent application Ser. No. 53,186 discloses a 48 amino acid immuno-modulatory protein, splenin, (hereafter referred to as "thysplenin") isolated from human spleen having the amino acid sequence -Gly-Leu-Pro-Lys-Glu-Val-Pro-Ala-Val-Leu-Thr-Lys-Gln-Lys-Leu-Lys-Ser-Glu-L eu-Val-Ala-Asn-Asn-Val-Thr-Leu-Pro-Ala-Gly-Glu-Met-Arg-Lys-Ala-Val-Tyr-Val- Glu-Leu-Tyr-Leu-Gln-Leu-Tyr-Leu-Gln-Ser-Leu-Thr-Ala-Glu-His-. Bovine thysplenin stimulates helper T cell activity in vivo in mice. Human thysplenin is thus expected to exhibit analogous biological activity in humans. Human thysplenin was described in the above-identified application as inducing elevation of intracellular cGMP in the human T cell line MOLT-4. The active site of bovine thysplenin, called SP-5, spans amino acid residues 32-36 thereof and has the sequence Arg-Lys-Glu-Val-Tyr. In application Ser. No. 56,186 the active site of the human sequence was disclosed as Arg-Lys-Ala-Val-Tyr. This application which issued as U.S. Pat. No. 4,923,964 (see also European Patent Application 292,302, published Nov. 23, 1988) is incorporated herein by reference.
Thysplenin, unlike thymopoietin, does not produce changes in biological activity of CEM cells. Thus thysplenin is implicated in the stimulation of T cell helper activity, not T cell suppressor activity. See also, for example, Goldstein, G. Nature (London) 247: 11-14 (1974); Basch, R. S. and Goldstein, G., Proc. Natl. Acad. Sci U.S.A., 71: 1474-1478 (1974); Scheid, M. P. et al J. Exp. Med.. 147: 1727-1743 (1978); Scheid, M. P. et al Science, 190: 1211-1213 (1975); Ranges, G. E. et al, J. Exp. Med., 156: 1057-1064 (1982); T. Audhya et al., Biochem, 20: 6195-6200 (1981); Venkatasubramanian, K., et al, Proc. Natl. Acad. Sci. U.S.A., 83: 3171-3174 (1986); Malaise M. G. et al, in "Immunoregulatory UCLA Symposium on Molecular and Cellular Biology", eds. Goldstein, G., et al (Liss, New York) (1986); Sunshine, G. H. et al, J. Immunol., 120: 1594-1599 (1978) and E. Rentz et al, Arch. Geschwulstforsch, 54(2): 113-118 (1948). See also U.S. Pat. Nos. 4,190,646; 4,261,886; 4,361,673; 4,420,424; and 4,629,723. Reference is made to the above-described patents, applications and articles for a discussion of other background material and the biological processes involved in the present invention.
U.S. Pat. No. 4,428,938 by Kisfaludy et al, issued Jan. 31, 1984, discloses certain peptides affecting immune regulation. Among such peptides are the following tetrapeptides:
Arg-Lys-Asp-Val PA1 Arg-Lys-Asn-Val PA1 Arg-Lys-Ala-Val PA1 Arg-Lys-Asp-Ala PA1 Arg-Lys-Asp-Ile PA1 Arg-Lys-Glu-Val PA1 Glp-Arg-Lys-Asp PA1 R is H, lower alkyl, acetyl, formyl, lower alkanoyl; PA1 X is Pro, dehydro-Pro, hydroxy-Pro, D-Lys, .alpha.-methylalanine (Aib), or Lys; PA1 Y is a D or L amino acid selected from Ala, Asp, Glu, Gln, Asn, beta Asp, Val, Leu, or Ile; PA1 Z is Gly or a D or L amino acid selected from Val, Ala, Leu or Ile; R.sup.1 is NR.sup.2 R.sup.3, wherein R.sup.2 and R.sup.3 are H or a straight chain or branched alkyl or alkenyl having 1 to 6 carbon atoms, optionally substituted with an aryl group or aryl substituted with either a halogen or a straight chain or branched alkyl or alkenyl having 1 to 6 carbon atoms or wherein R.sup.2 and R.sup.3 together comprise a cyclic methylene group of 3 to 7 carbon atoms, provided that when X is Lys, and Z is Val, Y is other than Asp, Asn, Ala, Asu, or Glu; and when X is Lys, Y is other than Asp; and when X is Ala and Z is Val, Y is other than Asp. PA1 R.sup.4 is H, lower alkyl, acetyl, formyl, lower alkanoyl, or des-amino; PA1 X' is Pro, dehydro-Pro, hydroxy-Pro, D-Lys, Aib, or Lys; PA1 Y' is a D or L amino acid selected from Val, Ile, Leu, Lys, Ala, Asp, Glu, Gln; PA1 Z' is a D or L amino acid selected from Tyr, Val, Leu, His, Ala, Phe or Trp; and PA1 R.sup.5 is OH or NR.sup.6 R.sup.7, wherein R.sup.6 and R.sup.7 are H or a straight chain or branched alkyl or alkenyl having 1 to 6 carbon atoms, optionally substituted with an aryl group or aryl substituted with either a halogen or a straight chain or branched alkyl or alkenyl having 1 to 6 carbon atoms or wherein R.sup.6 and R.sup.7 together comprise a cyclic methylene group of 3 to 7 carbon atoms.
The '938 patent generally includes the salts, amides, lower alkyl esters and protected derivatives of these sequences, as well as methods for using these sequences to treat immunological disorders due to thymic deficiencies. In this patent the peptides were tested for activity in an in vitro E rosette assay.
The same researchers reported such tetrapeptides in Kisfaludy et al, Hoppe-Seyler's Z. Physiol. Chem. B.D. 364, S. 933-940 (1983). In that paper it was reported that the sequence Arg-Lys-Glu-Val was a highly active analog in an in vitro E rosette test, and that the sequences Arg-Ala-Asp-Val and Arg-Lys-Ala-Val have drastically reduced activity.
There remains a need in the art for additional peptides which are useful in stimulating the immune system of humans for a variety of disease states to which deficiencies in T cell number or malfunctioning T cells contribute.